作者要感谢Fabrice Vincent对激光扫描共焦显微镜（LSCM）的支持，PhilippeBarrière和Thierry Blard进行每日超声卵巢扫描和hCG注射。这项工作部分得到"区域经济和区域伦巴第"项目"Ex Ovo Omnia"（AML授权号26096200）的支持。奖学金（2012年至2012年合约），FP7-PEOPLE-2011 CIG，研究执行机构（REA）"Pro-Ovum"（授予VL的第303640号）;"欧莱雅意大利"以及由欧洲社会基金，2007 - 2013年人力资源开发部门行动计划（合同编号:POSDRU / 89 / 1.5 / S / 62371至IM）共同资助的农业和兽医学博士后。 体内卵母细胞收集由Institut du Cheval et de l&#39;Equitation提供资金。

所有程序均经动物保护使用委员会CEEA Val de Loire No.19批准，并按照"实验动物护理和使用指导原则"进行。 卵母细胞收集和体外成熟<ol><li> 卵子接送 <ol><li>每日通过超声检查成年母体队列中卵巢卵泡的直径。在卵泡≥33mm（优势卵泡）出现时，在颈静脉上部注射1500IU人绒毛膜促性腺激素（hCG）的母马（iv）。 <ol><li>在进行注射前，用酒精拭拭该区域，找到颈静脉沟，并将拇指置于注射部位2-3厘米处，以引起静脉上升。将针几乎平行于颈部插入并抽出，以确保针在静脉中（血液将进入注射器）。此时，注射; hCG会诱导m优势卵泡卵母细胞的成熟。 </li></ol></li><li>注射hCG后35 h，用detomidine（15μg/ kg，iv），酒石酸丁醇（10μg/ kg，iv）和丁基东莨菪碱溴（0.2-0.3 mg / kg，15 mL / mare，iv）镇静母体。 </li><li>将针连接到超声波探头。将超声波探头插入阴道，并将卵巢经直肠对准超声探头。指示一名操作人员操纵针头，另一名操作者将卵巢固定在适当位置，使卵泡面向超声波探头。从具有优势卵泡的卵巢开始。 </li><li>用针穿刺阴道壁和优势卵泡壁（OPU的超声波探头配备有连接到抽吸系统的针的通道）;然后在回波图像中可见。 </li><li>将滤泡液吸入与抽吸系统连接的50mL锥形管中。倒入圆锥形内容物管成大型培养皿，并使用立体显微镜搜索卵丘卵母细胞复合物（COC）。 </li><li>由于COC并不总是与滤泡液一起回收，所以用含有5IU / mL肝素37℃的Dulbecco改性磷酸盐缓冲盐水（DPBS）冲洗滤泡。检查DPBS的COC存在，如先前在步骤1.1.5中针对滤泡液所述。冲洗数次直到检索到COC。 </li><li>一旦来自优势卵泡的COC被取回，穿刺并冲洗≥5和≤25mm的卵泡，如步骤1.1.3-1.1.4中对优势卵泡所述;卵泡≥5和≤25不表达促黄体激素/绒毛膜促性腺激素受体（LHCGR），因此，它们的卵母细胞不会响应于hCG而成熟，并且将在GV阶段（未成熟）收集。只保留COC与几层完整的卵丘细胞和棕色，细粒状的卵子。舍弃其他人</li><li>在OPU的最后，在用苄青霉素15000IU /动物注射母体以预防感染。 </li><li>将母马安置在一个安静，干净的稳定下至少2小时。通过确定耳朵的位置，对刺激的反应（ 例如噪音）和步态，在将母马返回其他动物的公司之前检查意识的迹象。 </li></ol></li><li> 体外成熟 <ol><li>补充有1,790IU / L肝素，0.4％牛血清白蛋白（BSA），青霉素和链霉素的HEPES缓冲TCM199（20mM Hepes）至37℃。提前准备这种介质。过滤消毒，并保持在4°C长达6个月。 </li><li>通过补充含有50ng / mL表皮生长因子（EGF）和20％小牛血清7的 NaHCO 3缓冲的TCM199（25mM NaHCO 3 ）来制备IVM培养基。打开新瓶后3周内使用NaHCO 3缓冲液TCM199。预先准备EGF作为100x股票，冻结在-20°C，并在6个月内使用。在4孔培养皿的孔中分配500μL，在38.5°C的5％CO 2的潮湿空气中孵育至少2小时。 </li><li>从≥5和≤25毫米卵泡中取出COC后，将其放入装有2 mL HEPES-TCM199的培养皿（直径3.5厘米）。将COC移至盘的不同区域，以清洗细胞碎片。 </li><li>将COC转移到IVM培养基中，在38.5℃的5％CO 2的加湿空气中孵育28小时。 </li></ol></li></ol> 2.免疫荧光和图像分析<ol><li> 染色体数 <ol><li>从优势卵泡或IVM结束收集后，在38.5°C的5％CO 2的潮湿空气中孵育具有100μMMonastrol的COC 1小时。预先准备monastrol作为100x股票，并将其存储在-20°C长达1年。 </li><li>去除卵丘细胞通过用0.5％透明质酸酶或轻轻移液处理它们;通过在0.2％的链霉蛋白酶处理它去除透明带。预先将透明质酸酶和链霉蛋白酶预先作为10x储备，并将其储存在-20°C长达1年。 </li><li>将DPBS中的4％多聚甲醛中的卵母细胞在38.5℃下固定15分钟，然后在4℃下固定45分钟。 警告！处理多聚甲醛时请戴上个人防护装备，并按照危险废物处置指南处理受污染的物质。 </li><li>通过在填充有500μL补充有0.1％聚乙烯醇（PVA）的DPBS的3孔中顺序转移来洗涤卵母细胞。在室温（RT）下，在0.3％Triton X-100中渗透10分钟。 </li><li>在加入1％牛血清白蛋白（DPBS-1％BSA）和10％正常驴血清的DPBS中，在室温下封闭非特异性结合孵育至少30分钟。样品可以在4℃下保持封闭溶液3-4天。 </li><li>对于原代染色，在4℃下补充有1％BSA（稀释倍数1:50）的DPBS中，将兔抗Aurora B磷酸-323-2卵母细胞孵育过夜。 </li><li>如步骤2.1.4所述洗涤卵母细胞。将卵母细胞在黑色中在室温下在异硫氰酸四甲基罗丹明（TRITC） - 缀合的驴抗兔IgG（DPBS-1％BSA中1:100）的溶液中孵育1小时。 </li><li>按步骤2.1.4中所述清洗卵母细胞，然后将3-4个卵母细胞放入一滴不含硬化的抗褪色载体中，补充有20μMYOPRO1载玻片。重复上述步骤，直到所有的卵母细胞被安装（每个载玻片3-4个卵母细胞）。 </li><li>让卵母细胞在载物介质中沉降约10分钟，然后用盖玻片盖住它们。为了避免粉碎卵母细胞，在将玻片放在玻璃片上之前应用两条双面胶带。 注意:此预防措施还将确保所有样品在玻璃are之间均匀压缩de和盖玻片。玻璃载玻片可以在-20℃冷冻，并在随后的2-3天内成像。 </li><li>使用红色（着丝粒）和绿色（染色体）通道7,13,14,20,21，用60X物镜在共聚焦激光扫描显微镜上成像样品。在z轴上扫描整个异位板的样品，每0.35μm的步长。保存每个计划的数字化图像。 </li><li>使用NIH ImageJ软件的细胞计数功能（可从https://imagej.nih.gov/ij/plugins/cell-counter.html获得）计数串联共焦区域中的着丝点。 注意:避免重复计数出现在相邻部分上的着丝粒，通过识别出现，停留和使用数字代码在连续部分消失的着丝粒。 </li><li>重复染色体共在步骤2.1.11中与独立操作员进行通信。 注意:将卵母细胞分类为整倍体（32条染色体），超倍体（&gt; 32）或次倍体（&lt;32）。 </li></ol></li><li> 主轴形态和组蛋白乙酰化 <ol><li>从优势卵泡或IVM结束后收集，通过在0.5％透明质酸酶处理或通过温和移液去除卵丘细胞，如步骤2.1.2所述。 </li><li>洗涤DPBS-PVA中的卵母细胞，如步骤2.1.4，并在38℃下将它们固定在2.5％多聚甲醛中20分钟。如步骤2.1.4所述进行广泛洗涤，并在室温下在0.1％Triton X-100中透化5分钟。 注意:处理多聚甲醛时应戴上个人防护装备，并按照危险废物处理指南处理受污染的物质。 </li><li>在补充有2％牛血清白蛋白（DPBS-2％BSA），0.05％皂苷和10％正常驴血清的DPBS中阻断非特异性结合孵育r 2 h。 注意:样品可以在4℃下保存在封闭溶液中3-4天。 </li><li>对于原代染色，在含有0.05％皂角苷的DPBS-2％BSA中，在4℃孵育小鼠抗-α-微管蛋白（1:150）中的卵母细胞和兔抗acH4K16（1:250）过夜。 </li><li>洗涤卵母细胞如步骤2.1.4。在含有0.05％皂苷的DPBS-2％BSA中的绿色荧光染料缀合的驴抗小鼠IgG（1:500）和TRITC缀合的驴抗兔IgG（1:100）的溶液中孵育卵母细胞1小时RT在黑暗中。 </li><li>按步骤2.1.4清洗卵母细胞，然后将3-4个卵母细胞置于一滴补充有1μg/ mL 4&#39;，6-二脒基-2-苯基吲哚（DAPI）的非硬化抗褪色载体中，在玻璃片上。 </li><li>将载玻片上的卵母细胞载于步骤2.1.8。 </li><li>使用红色（acH4K16），绿色（α-微管蛋白）和蓝色（DNA）通道的60x物镜在共聚焦激光扫描显微镜上成像样品。 注意:在采集所有样品时，请保持红色和蓝色通道的激光设置不变。扫描整个减数分裂主轴和中间板在z轴上的样品，每0.35微米的步长。保存数字化图像（单图和投影3D图像）。 </li><li>使用NIH ImageJ软件的测量功能（https://imagej.nih.gov/ij/docs/guide/146-30）测量投影图像上最大宽度的极点主轴长度和主轴直径的.html）。重复3次，计算平均值。 </li><li>使用NIH ImageJ软件的集成密度函数（https://imagej.nih.gov/ij/docs/guide/146-30.html）量化acH4K16的相对荧光。将其归一化为DAPI染色的整合密度。 </li></ol></li></ol> 3. mRNA表达分析<ol><li>在从5至25毫米卵泡中取出COCs之后，收集颗粒细胞维持在培养皿中，并在冷DPBS中以10,600 xg离心2分钟洗涤两次。在液氮中快速冻结。将样品储存在-80°C长达6个月;细胞将用于标准曲线。 </li><li>仔细地从未成熟（GV）， 体外成熟和体内成熟的卵母细胞中去除所有卵丘细胞，如步骤2.1.2所述。 </li><li>洗涤卵母细胞，如2.1.4所述，在DPBS-PVA中，以1μL的体积单独收集，并在上面放置2μLRNALater。在液氮中快速冷冻。将样品储存在-80°C达6个月。 </li><li>向每个样品添加2 pg 萤光素酶RNA作为穗状。将卵母细胞2×2收集，并使用适合从小样品回收RNA的基于二氧化硅膜滤器的试剂盒提取总RNA。 </li><li>用0.25μg随机六聚体和小鼠Moloney白血病病毒逆转录酶在10μL反转录RNA 1 h，37＆＃<html> 176℃。将样品在-20°C下储存长达6个月。 </li><li>将无RNAse水的颗粒细胞中的cDNA稀释至50 ng /μL。连续稀释该溶液1:10，得到5 ng /μL，0.5 ng /μL，0.05 ng /μL和0.005 ng /μL溶液。 </li><li>使用相当于0.05个卵母细胞作为底物的cDNA，或者5μL连续稀释的颗粒细胞cDNA，10μL的SYBR绿色超级细胞和0.3μM的每种特异性引物（每个反应物表1 ）。 </li><li>使用3步方案在热循环仪中运行反应:95℃30秒，60℃30秒，72℃20秒，重复40次。最后，从60°C开始，获得熔融曲线，每20s高达95°C将温度提高0.5°C。 </li><li>使用标准曲线评估卵母细胞样品中特异性mRNA的起始量（SQ）基于颗粒细胞样品计算，将数据表示为感兴趣基因的SQ和持家基因的SQ之间的比例。 </li></ol>

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作者没有什么可以披露的。

即使IVM已经在马上进行了超过二十年16 ，我们还不知道卵母细胞是否可以是胚胎非整倍体的起源，正如人类提出的一样。原因可能是卵母细胞扩散用于染色体计数导致相当大的样品损失。考虑到这一点，进行了调查染色体分离错误的方法的调查，以寻找适用于马卵母细胞的最合适的技术。在科学文献中发现了四种主要技术:1）染色体扩增5,18,19,2）荧光原位杂交（FISH）4,6,17,20,3）染色体计数在monastrol-折叠的心轴上23,24,25 。 如已经提到的，染色体扩散的随后的吉姆萨染色的制备由于高的样品损失率而变得复杂。在许多情况下，这种方法已被FISH所替代，FISH使用特定的探针来映射不同的染色体。 FISH技术的缺点是当使用少量探针时，假阴性的发生率可能增加（ 即，鉴定为非整倍体的细胞，因为缺失或超编号染色体尚未被探测）。因此，FISH的可靠使用主要是基于更常见于非整倍体影响的染色体的先验知识（ 例如人，21号染色体）。以前，FISH已经用于研究使用2个探针，针对染色体2和染色体4 20的马胚胎染色体异常。根据这些实验， 体外产生的胚胎的核比体内产生的胚胎更可能受到染色体异常的影响。该发现与上述使用IVM卵母细胞中的monastrol-collapse方法的观察结果一致。然而，IVM卵母细胞中非整倍体的发生率甚至高于在体外产生的胚胎20中使用FISH报告的发生率。这种部分差异可以通过仅使用两种染色体特异性探针用于FISH实验的事实来解释。这种方法可能低估了涉及其他染色体的非整倍体的发生率，特别是考虑到马有32条染色体时。然而，它不能被排除在严重的动作之外卵母细胞中可检测到的倍性不保留在胚胎中，因为受关键遗传异常影响的配子可能不会进一步发展。 monastrol治疗，染色体和着丝粒染色和共焦显微镜的组合允许在MII阶段马卵母细胞中染色体数量的一致计数，具有最小的样品损失。在开发这种马卵母细胞技术方面遇到的技术限制是马特异性抗体的可获得性。在使用Aurora B磷酸（Thr232）之前，另外两种抗体（Anti-Aurora B和anti-CENPA）不产生着丝粒染色。值得注意的是，抗Aurora B和抗CENPA已经成功地用于牛和人类细胞26,27,28 ;因此，得出结论，他们不是马的具体。这种技术也可能受到共聚焦激光器sc的可用性的限制anning显微镜和图像分析软件。此外，由两个独立运算符进行盲计数是排除运算符引起的伪像的关键。即使一个monastrol折叠的纺锤体的染色体计数是更好地保留细胞形态并防止样品丢失的方法，该技术仅允许观察染色体基本数量的差异;对于其他染色体异常（ 例如易位，分段交换等 ），可以通过核型分析，在某些情况下，通过FISH来显示它。 染色体分离的错误也通过纺锤体和染色体免疫荧光进行了研究，而不进行染色体计数23,24,25 。在这种情况下，具有严重异常和滞后或分散染色体的心轴被认为是不规则色素的迹象我隔离因此， 在体外成熟的卵母细胞7 （更可能受非整倍体影响的类别）中观察到离开纺锤体的染色体。然而，染色体计数具有传递可量化信息的优点。 总而言之，与其他所描述的技术相比，使用monastrol和着丝粒染色具有防止样品损失，最小化假阴性的发生率和可量化的优点。 已经提出了表观遗传修饰的改变作为卵母细胞非整倍体发生的可能机制18,24,25。使用三重荧光染色，可以可视化和测量减数分裂纺锤体并观察滞后/分散的染色体。同时，感谢抗体的发展识别特异性残基修饰，在相同的样品上进行H4K16的乙酰化水平。该方法具有减少分析所需的卵母细胞数量和将H4乙酰化与主轴形态相关的双重目的。 共价蛋白质修饰的定量可以通过Western印迹进行。然而，该技术需要更大的样品量，并且不保留样品的形态学研究的完整性。三重荧光染色方法可以应用于其他组蛋白修饰，并且可能应用于可被不同二级抗体8识别的任何蛋白质;唯一的限制因素是马特异性抗体的可用性。在这方面，作者希望提高对马模型的关注将增加对这一物种基本生物学机制调查的兴趣，因此在发展中专用工具。 最后，还描述了通过逆转录和定量PCR（q-PCR）进行的编码组蛋白脱乙酰酶（HDAC）和乙酰转移酶（HAT）的mRNA的表达研究。 q-PCR是广泛传播的技术;然而，它对马卵母细胞的使用并不扩散。必须指出，由于在卵母细胞成熟期间转录活性的普遍沉默29 ，总转录物量的分析只能在这个背景30,31中揭示mRNA的稳定性或降解。没有观察到体外和体内成熟期间转录本的相对表达的差异。这些研究结果表明，翻译和翻译后法规可能受到IVM的影响，指出需要开发其他旨在调查翻译和po的实验方法单细胞水平的单一翻译机制。 总体而言，本研究描述了一种用于形态和生物化学表征马卵母细胞的实验方法。这种方法可以应用于同样受低回收率影响的其他物种的卵母细胞，包括人类或濒危物种。这些研究将扩大我们对哺乳动物生殖生物学的知识，并将有助于我们对不育症的理解。

已经开发了广泛的辅助生殖技术来治疗妇女，伴侣动物和濒危物种的不育症。临床设置中最常见的手术之一是通过超声引导经阴道抽吸，卵子吸收（OPU） 1从卵巢卵泡中检索中期II（MII）阶段卵母细胞。然后将这些卵母细胞体外受精 （IVF），将所得的胚胎植入受体子宫。在外源性促性腺激素给药后，取出MII期（成熟）卵母细胞。然而，这种治疗在一些患者中与卵巢过度刺激综合征（OHSS）的发展有关2 。 利用完全成熟的未成熟卵母细胞（GV-stage）的内在能力自发恢复减数分裂，从卵泡中分离出来，可以获得成熟的卵母细胞，而不需要管理调理促性腺激素3 。该过程称为卵母细胞体外成熟（IVM），并且代表了辅助生殖技术的药物导向较少，成本更低且更耐用的方法。然而，胚胎发育与体外成熟卵母细胞的成功通常低于体内成熟卵母细胞4,5 。可能的解释是， 体外成熟的卵母细胞更多地受到染色体分离中的错误的影响，并且由此产生的非整倍体损害了正常的胚胎发育6 。 了解IVM期间染色体错分离的分子基础将最终披露这种技术的全部潜力。在这方面，实验方法用于研究体外成熟卵母细胞的形态和生化特征， 与体内成熟相比这里描述了卵母细胞7,8 。具体来说，使用成年和自然循环马作为实验模型说明未成熟卵母细胞的OPU和未成熟卵母细胞的IVM的程序。然后，使用免疫荧光和图像分析来研究染色体分离，纺锤形态和组蛋白乙酰化对这些配子的全局模式。最后，描述了用于mRNA表达分析的逆转录和定量PCR方案。 与啮齿动物模型相比，马不允许遗传操作，操作不太方便，需要昂贵的维护。然而，由于与人类卵巢生理学的相似性，这种模型对于卵母细胞成熟的研究正在获得相当大的兴趣，/ SUP&gt;。此外，在马中制定IVM-IVF的可靠方案具有重大的经济利益，因为它将允许从遗传价值高的母马的数量增加。 对卵母细胞进行实验的一个局限性，特别是在单调节物种中，是限制样品的可用性。通过将以前在小鼠中开发的方法调整到马卵母细胞13,14以便进行最小化样品损失的染色体计数（参见与其他可用技术的比较的讨论），已经克服了这个限制。此外，已经优化了三重荧光染色方案以对相同的样品进行多重分析，并且仅在2个卵母细胞池上进行q-PCR分析。 总体而言，本研究描述了一种旨在形态和生物化学方面的实验方法使马卵母细胞变得更细，这种细胞类型由于样本可用性低而极具挑战性。然而，它可以扩大我们对生殖生物学和不育性不育的知识。

<table border="0" cellpadding="0" cellspacing="0" width="802">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:369px;">ultrasound probe</td>
			<td style="width:159px;">Aloka</td>
			<td style="width:113px;">UST-5820-7,5</td>
			<td style="width:161px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human chorionic gonadotrophin&#160;</td>
			<td>centravet</td>
			<td>CHO004</td>
			<td>1500unit/animal IV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">detomidine</td>
			<td>centravet</td>
			<td>MED010</td>
			<td>9-15&#181;g/kg IV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">butylscopolamine bromure&#160;</td>
			<td>centravet</td>
			<td>EST001</td>
			<td>0,2mg/kg IV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">stereomicroscope</td>
			<td style="width:159px;">NIKON</td>
			<td style="width:113px;">SMZ-2B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">butorphanol</td>
			<td>centravet</td>
			<td>DOL003</td>
			<td>10&#181;g/kg IV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">benzyl-penicillin</td>
			<td>centravet</td>
			<td>DEP203 IM</td>
			<td>15000UI/animal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TCM199</td>
			<td>Sigma-Aldrich</td>
			<td>M3769 10X1L</td>
			<td>powder for hepes-buffered TCM199</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hepes sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>H3784-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A8806</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">heparin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>H3149-10KU</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaHCO3-buffered TCM199</td>
			<td>Sigma-Aldrich</td>
			<td>M2154-500ML</td>
			<td>liquid for IVM medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">epidermal growth factor</td>
			<td>Sigma-Aldrich</td>
			<td>E4127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">newborn calf serum</td>
			<td>Sigma-Aldrich</td>
			<td>N4762-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-well dishes</td>
			<td>NUNCLON</td>
			<td>144444</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">incubator</td>
			<td>Heraeus</td>
			<td>BB6060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">monastrol</td>
			<td>Sigma-Aldrich</td>
			<td>M8515</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hyaluronidase</td>
			<td>Sigma-Aldrich</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pronase</td>
			<td>Sigma-Aldrich</td>
			<td>P5147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polyvinyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>341584</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">triton-X 100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">normal donkey serum&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rabbit anti-Aurora B phospho-Thr232&#160;</td>
			<td>BioLegend</td>
			<td>636102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRITC-conjugated donkey anti-rabbit IgG&#160;</td>
			<td>Vector Laboratories</td>
			<td>711-025-152</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vecta-Shield</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">YOPRO1</td>
			<td>Thermofisher Scientific</td>
			<td>Y3603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">confocal laser scanning microscope&#160;</td>
			<td>LSM 780&#160;</td>
			<td>Zeiss</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">confocal laser scanning microscope&#160;</td>
			<td>LSM 700&#160;</td>
			<td>Zeiss</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ software</td>
			<td colspan="2">rsb.info. nih.gov/ij/download.html&#160;</td>
			<td>free resource</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse anti-alpha-tubulin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T8203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rabbit anti-acH4K16</td>
			<td>Upstate Biotechnology</td>
			<td>07-329</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AlexaFluor 488-coniugated donkey anti-mouse IgG</td>
			<td>Life Technologies</td>
			<td>A21202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#8217;,6-diamidino-2-phenylindole&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D8417</td>
			<td>DAPI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">centrifuge</td>
			<td>Eppendorf</td>
			<td>5417R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNALater</td>
			<td>Invitrogen</td>
			<td>AM7020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luciferase RNA</td>
			<td>Promega</td>
			<td>L4561</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PicoPure RNA Isolation Kit&#160;</td>
			<td>Applied Biosystems</td>
			<td>12204-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">random hexamers&#160;</td>
			<td>Thermofisher Scientific</td>
			<td>N8080127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse Moloney leukaemia virus reverse transcriptase</td>
			<td>Thermofisher Scientific</td>
			<td>28025013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYBR green supermix&#160;</td>
			<td>BioRad</td>
			<td>1708880</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">specific primers</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>specific primers were designed using Primer3Plus software (free resource)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">thermal-cycler&#160;</td>
			<td>BioRad</td>
			<td>MyiQ&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse monoclonal anti-CENPA</td>
			<td>Abcam</td>
			<td>ab13939</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse monoclonal anti-Aurora B</td>
			<td>Abcam</td>
			<td>ab3609</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of chromosome segregation, histone acetylation, and spindle morphology in horse oocytes

辅助生殖领域已被开发用于治疗妇女，伴侣动物和濒危物种的不育症。在马上，辅助生殖也允许从高绩效者生产胚胎，而不会中断他们的运动生涯，并有助于增加从高遗传价值的母马的数量。本手稿描述了使用卵子吸收（OPU）从马卵巢收集未成熟和成熟卵母细胞的程序。然后通过适应以前在小鼠中发展的方案，将这些卵母细胞用于研究非整倍体的发生率。具体而言，共焦激光显微镜扫描后，染色体和中期II（MII）卵母细胞的着丝粒被荧光标记并计数在连续的焦点图上。该分析显示，当从卵泡中收集未成熟卵母细胞并在体外成熟时，非整倍体率的发生率较高在体内。微管蛋白的免疫染色和特定赖氨酸残基的组蛋白4的乙酰化形式也揭示了减数分裂纺锤体的形态和组蛋白乙酰化的全局模式的差异。最后，通过逆转录和定量PCR（q-PCR）研究了编码组蛋白脱乙酰酶（HDAC）和乙酰转移酶（HAT）的mRNA的表达。 在体外和体内成熟的卵母细胞之间没有观察到转录物的相对表达的差异。与卵母细胞成熟期间转录活性的一般沉默一致，总转录物量的分析只能揭示mRNA的稳定性或降解。因此，这些研究结果表明，其他翻译和翻译后法规可能会受到影响。 总体而言，本研究描述了一种用于形态和生物化学表征马卵母细胞的实验方法由于样品可用性低，这种类型的研究极具挑战性。然而，它可以扩大我们对单调繁殖生物学和不孕症的知识。

这些实验的原始发现在以前已经深入描述7并且在本文中作为可以使用所述方案获得的结果的实例进行报道。 成熟率在OPU从优势卵泡获得的32个COCs中，28个（88％）处于MII阶段。在5-25mm大小的卵泡中收集的58个COC中的14个被立即快速冷冻为未成熟的卵母细胞用于mRNA表达研究。其余44例体外成熟，37例达到MII期（85％）。两组成熟效率差异无统计学意义（Fisher精确检验P&gt; 0.05）。 非整倍体率为了研究IVM can扰乱了次级卵母细胞和第一极体之间的忠实染色体分配，计数了MII期染色体的数量，因为它代表减数分裂期间染色体分离的结果I. 如图1A所示 ，抗Aurora B磷酸Thr232抗体特异性染色马卵母细胞中的着丝粒区域，允许人们对染色体数进行计数。 体外成熟卵母细胞与体内成熟卵母细胞（0/12; Fisher精确检验，P &lt;0.05; 图1B ）相比，非整倍体（5/11）显着受影响。大多数非整倍体卵母细胞（4/5）均为超倍体;因此，与染色体扩散一样，非整倍体率不是由于确定染色体损失的样品的制备中的假象产生的。还尝试使用抗CENPA和抗AURKB抗体的着丝粒区域的免疫染色，但没有信号在这两种情况下都是这样的（数据未显示）。 H4K16的主轴形态和乙酰化 如图2所示 ，测量极 - 极主轴长度和主轴最大宽度的直径。根据这些测量，与体内成熟卵母细胞相比，体外成熟卵母细胞（19.89±1.15μm）和更宽（17.28±0.81μm）（长度为14.57±1.7μm，直径为13.56±0.91μm;未配对t -test，P &lt;0.05）。在相同的样品上，也测量了H4K16的全球乙酰化水平，证实了与体内对应物相比，IVM卵母细胞中H4K16的乙酰化显着降低（ 图2 ，红色）。 表达式编码组蛋白乙酰转移酶和脱乙酰酶的脚本通过q-PCR 研究体外成熟卵母细胞中参与组蛋白乙酰化 - 脱乙酰化过程的基因表达是否发生改变。组蛋白乙酰转移酶1（ HAT1 ），K-乙酰转移酶8 （KAT8 ），组蛋白脱乙酰酶1（ HDAC1 ）和NAD依赖蛋白脱乙酰酶sirtuin 1（ SIRT1 ）被确定为推定靶标。在未成熟， 体外成熟和体内成熟的卵母细胞中这些转录物的表达的显着差异没有观察到，与使用的归一化无关。具体来说，使用SQ方法，使用标准曲线计算图3中报道的转录物表达分析的结果，并对归属于甘油醛-3-磷酸脱氢酶（ GAPDH ）进行归一化。同样，没有diffe当对荧光素酶的尖峰进行归一化的结果或当使用ΔΔct方法（未示出）时，观察到肾脏。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 目标 </td><td> Fw底漆 </td><td> Rv引物 </td><td> 放大镜尺寸 </td></tr><tr><td> GAPDH </td><td> ATCACCATCTTCCAGGAGCGAGA </td><td> GTCTTCTGGGTGGCAGTGATGG </td><td> 241 bp </td></tr><tr><td> HAT1 </td><td> CTGACATGAGTGATGCTGAACA </td><td> TAACGCGTCGGTAATCTTCC </td><td> 219 bp </td></tr><tr><td> HDAC1 </td><td> GAAGGCGGTCGCAAGAAT </td><td> CCAACTTGACCTCCTCCTTGA </td><td> 166 bp </td></tr><tr><td> KAT8 </td><td> ACTGGTCTTGGGTCCTGCTG </td><td> GGGCACTTTTGAGGTGTTCC </td><td> 198 bp </td></tr><tr><td> 荧光素酶 </td><td> TCATTCTTCGCCAAAAGCACTCTG </t<html> D&gt; <td> AGCCCATATCCTTGTCGTATCCC </td><td> 148 bp </td></tr><tr><td> SIRT1 </td><td> GACTCGCAAAGGAGCAGATT </td><td> GGACTCTGGCATGTTCCACT </td><td> 169 bp </td></tr></tbody></table> 表1:引物设置。对于分析的每个转录物，给出正向（Fw）和（Rv）引物的5&#39;&gt; 3&#39;序列和预期的扩增子大小:甘油醛-3-磷酸脱氢酶（ GAPDH ） ，组蛋白乙酰转移酶1（ HAT1 ） ，组蛋白脱乙酰酶1（ HDAC1 ） ， K-乙酰转移酶8（ KAT8 ） ，荧光素酶和NAD依赖蛋白脱乙酰酶sirtuin 1（ SIRT1 ）。 转载自参考文献7的许可。 /55242fig1.jpg"/&gt; 图1:体内和体外马卵母细胞中非整倍 体率。 （ A ）显示用monastrol处理的MII阶段马卵母细胞的极光B磷酸 - Thr232（红色）和DNA（绿色）染色的代表性图像。显示了整倍体卵母细胞（32条染色体）。刻度棒=5μm。 （ B ）条形图表示体内 （n = 12）和体外 （n = 11）成熟卵母细胞中整倍体和非整倍体MII阶段卵母细胞的分布 。 *表示非整倍体率有显着性差异（Fisher精确检验，P &lt;0.05）。转载自"参考文献7 <a href="http://ecsource.jove.com/files/ftp_upload/55242/55242fig1large.jpg" target="_blank">"的</a>许可。 <a href="http://ecsource.jove.com/files/ftp_upload/55242/55242fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg"/ src ="/ files / ftp_upload / 55242 / 55242fig2.jpg"/&gt; 图2:体内和体外马卵母细胞中的主轴测量和H4K16乙酰化。 在体内 （n = 4）或体外 （n = 14）成熟后，在MII卵母细胞中显示微管蛋白（绿色），乙酰化H4K16（红色）和DNA（蓝色）的代表性图像。微管图像显示用于主轴长度和直径测量的轴。刻度棒=5μm。参考文献7修改。 <a href="http://ecsource.jove.com/files/ftp_upload/55242/55242fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55242/55242fig3.jpg" /> 图3:KAT8， SIRT1 ， HAT1 ， 和HDAC1在Immature， In Vivo ， 和体外成熟的卵母细胞。条形图表示KAT8， SIRT1 ， HAT1的相对mRNA表达的平均值±SEM， 和HDAC1 ， 表示为与用作家务的GAPDH的SQ相比，感兴趣的抄本的起始数量（SQ）。未成熟（GV，n = 14）， 体内 （n = 14）和体外 （n = 12）成熟卵母细胞之间无统计学差异（单因素方差分析， P &gt; 0.05）。转载自参考文献7的许可。 <a href="http://ecsource.jove.com/files/ftp_upload/55242/55242fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes at JoVE.com

马卵母细胞染色体分离、组蛋白乙酰化和纺锤体形态分析- 中国优秀硕士学位论文全文数据库发育生物学|视频

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

该手稿描述了一种用于形态和生物化学表征马卵母细胞的实验方法。具体来说，本文通过超声引导的卵子吸收（OPU）如何收集不成熟和成熟的马卵母细胞，以及如何调查染色体分离，纺锤形态，全局组蛋白乙酰化和mRNA表达。

马卵母细胞染色体分离，组蛋白乙酰化和主轴形态分析

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted ...

analysis-chromosome-segregation-histone-acetylation-spindle

Nanyang Technological University

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SCIENTISTS IN ACTION

10.9K 次观看.  University of Milan. 该手稿描述了一种用于形态和生物化学表征马卵母细胞的实验方法。具体来说，本文通过超声引导的卵子吸收（OPU）如何收集不成熟和成熟的马卵母细胞，以及如何调查染色体分离，纺锤形态，全局组蛋白乙酰化和mRNA表达。 

视频: Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

荧光蛋白的表达<em&gt;披文昌鱼</em&gt;通过基因注射到未受精的卵母细胞

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

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发育生物学

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded Nucleolus) or unable (NSN, Not Surrounded Nucleolus) to develop to the blastocyst stage after in vitro maturation (IVM) and in vitro fertilization (IVF). It makes use of Hoeschst33342 (or any other DNA intercalating dye) able to bind to the heterochromatin of the nucleolus showing a ring in the SN oocytes or not, like in the NSN oocytes. This represents the easiest and quickest way to sort both antral oocytes that can be eventually used for IVM or IVF procedures. 
Briefly, the protocol consists of the following steps: hormone injection to stimulate follicular growth; isolation of the oocytes at the GV stage from the antral compartment by puncturing the ovary with a sterile needle; preparation of thin glass pipettes for mouth pipetting of the oocytes; sorting of the oocytes with Hoechst33342 prepared at a supravital concentration; IVM, IVF or any other molecular/cellular analysis.
Unfortunately there are still few evidences to sort SN and NSN oocytes using less invasive techniques. If and once they will be identified, they could be potentially applied to human assisted reproductive technologies, although with several aspects that should be modified. To date, this technique has potential implications to dramatically increase IVM and IVF successful procedures in both endangered and species with economic interest.

Here we present a protocol for the characterization of mouse antral oocytes based on nucleolar organization.

基于核仁染色质组织分离鼠标窦卵母细胞与表征

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

在Active ERK的时空分析<em&gt;℃。线虫</em&gt;生殖系

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

通过显微注射卵母细胞生成转基因小鼠

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

有丝分裂过程中染色体分离的活体细胞成像

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Visualization and Analysis of Pharyngeal Arch Arteries using Whole-mount Immunohistochemistry and 3D Reconstruction

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart disease. To study the formation of PAAs, we developed a protocol using whole-mount immunofluorescence coupled with benzyl alcohol/benzyl benzoate (BABB) tissue clearing, and confocal microscopy. This allows for the visualization of the pharyngeal arch endothelium at a fine cellular resolution as well as the 3D connectivity of the vasculature. Using software, we have established a protocol to quantify the number of endothelial cells (ECs) in PAAs, as well as the number of ECs within the vascular plexus surrounding the PAAs within pharyngeal arches 3, 4, and 6. When applied to the whole embryo, this methodology provides a comprehensive visualization and quantitative analysis of embryonic vasculature.

Here, we describe a protocol to visualize and analyze the pharyngeal arch arteries 3, 4, and 6 of mouse embryos using whole-mount immunofluorescence, tissue clearing, confocal microscopy, and 3D reconstruction.

使用全载免疫作用化学和三维重建对咽弓动脉的可视化与分析

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart ...

Minimum Volume Vitrification of Immature Feline Oocytes

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to promote biodiversity preservation. In felids, most species are threatened with extinction, and domestic breeds are used as a model to increase the efficiency of protocols for germplasm banking. Among oocyte cryopreservation techniques, vitrification is more and more popular in human and veterinary assisted reproduction. Cryotop vitrification, which was at first developed for human oocytes and embryos, has demonstrated to be well-suited for cat oocytes. This method offers several advantages, such as the feasibility in field conditions and the speed of the procedure, which can be helpful when several samples need to be processed. However, the efficiency is strongly dependent on the operator&#8217;s skills, and intra- and inter-laboratory standardization are needed, as well as personnel training. This protocol describes minimum volume vitrification of immature feline oocytes on a commercial support in a step by step field-friendly protocol, from oocyte collection to warming. Following the protocol, preservation of oocyte integrity and viability at warming (as high as 90%) can be expected, although there is still room for improvement in post-warming maturation and embryonic development outcomes.

This manuscript describes a protocol for the minimum volume vitrification of immature cat oocytes with laboratory-made media on commercial supports. It covers every step from oocyte isolation from ex vivo gonads to vitrification and warming.

未成熟费林卵母细胞的最小体积玻璃化

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to ...

In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that during the reproductive life span only about 1% of the oocytes in an ovary mature and ovulate, several techniques have been developed to increase the exploitation of the ovarian reserve to the growing population of non-ovulatory follicles. Such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. However, the vast potential of the ovarian reserve is still largely unexploited. In cows, for instance, some attempts have been made to support in vitro culture of oocytes at specific developmental stages, but efficient and reliable protocols have not yet been developed. Here we describe a culture system that reproduce the physiological conditions of the corresponding follicular stage, defined to develop in vitro growing oocytes collected from bovine early antral follicles to the fully-grown stage, corresponding to the medium antral follicle in vivo. A combination of hormones and a phosphodiesterase 3 inhibitor was used to prevent untimely meiotic resumption and to guide oocyte&#39;s differentiation.

We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage. &#160;

牛早期蚁卵泡卵母细胞的体外培养策略

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that ...

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of oocytes and surrounding follicular cells. It is vital to separate both follicular cells and oocytes for various research purposes such as for primary culture of follicular cells, analysis of gene expression, oocyte maturation and in vitro fertilization, etc. The conventional method uses forceps to separate both compartments, which is laborious, time consuming and has high damage to the oocyte. Here, we have established a simple method to separate both compartments using a pulled glass capillary. Under a stereomicroscope, oocytes and follicular cells can be easily separated by pipetting in a pulled fine glass capillary (the diameter depends on the follicle diameter). Compared with the conventional method, this new method has high efficiency in separating both oocytes and follicular cells and has low damage to the oocytes. More importantly, this method can be applied to early-stage follicles including at the pre-vitellogenesis stage. Thus, this simple method can be used to separate follicular cells and oocytes of zebrafish.

Here, we present a simple method for separating follicular cells and oocytes in zebrafish ovarian follicles, which will facilitate investigations of ovarian development in zebrafish.

斑马鱼卵巢卵巢中卵泡细胞和卵母细胞的分离

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of ...

马卵母细胞染色体分离，组蛋白乙酰化和主轴形态分析

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